Non-steroidal farnesoid x receptor modulators

ABSTRACT

The efficient regulation of cholesterol synthesis, metabolism, acquisition, and transport is an essential component of lipid homeostasis. The farnesoid X receptor (FXR) is a transcriptional sensor for bile acids, the primary product of cholesterol metabolism. Accordingly, the development of potent, selective, small molecule agonists, partial agonists, and antagonists of FXR would be an important step in further deconvoluting FXR physiology. In accordance with the present invention, the identification of novel potent FXR activators is described. Two derivatives of invention compounds, bearing stilbene or biaryl moieties, contain members that are the most potent FXR agonists reported to date in cell-based assays. These compounds are useful as chemical tools to further define the physiological role of FXR as well as therapeutic leads for the treatment of diseases linked to cholesterol, bile acids and their metabolism and homeostasis.

FIELD OF THEE IVENTION

The present invention relates to new chemical entities. In a particular aspect, the present invention relates to non-steroidal modulators of farnesoid X receptors (FXR). In another aspect, the present invention relates to methods for modulating FXR-mediated processes employing the novel compounds described herein.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.

The efficient regulation of cholesterol biosynthesis, metabolism, acquisition and transport is an essential function of mammalian cells. High levels of cholesterol are associated with atherosclerosis, a leading cause of death in the western world and a major risk factor correlated with the occurrence of coronary heart disease and stroke. Until recently, recommendations for the treatment of hypercholestemia were focused on the use of statins, which inhibit the de novo biosynthesis of cholesterol, and the use of bile acid sequestering agents. While statin-based agents are still in widespread use as cholesterol-lowering drugs, an evolving understanding of the mechanisms controlling cholesterol homeostasis has led to new molecular targets as candidates in therapeutic intervention.

Cholesterol metabolism is controlled through a complex feedback loop involving cholesterol itself and bile acids (which are primary oxidation products), and through secretion in the gut, the single most critical regulators of cholesterol absorption. The nuclear receptors LXR (liver X receptor) and FXR (farnesoid X receptor) are the specialized sensors of cholesterol and bile acids that control transcription of networks encoding key metabolic enzymes. For example activation of LXR by oxysterols (i.e., mono-oxygenated cholesterol metabolites) leads to the up-regulation of CYP7A1, the enzyme that catalyzes the rate limiting step in the conversion of cholesterol to bile acids. In turn, bile acids such as chenodeoxycholic acid (CDCA, 1, a low affinity endogenous agonist for FXR, whose structure is shown below) are potent ligands for FXR, whose activation leads to down-regulation of CYP7A1, leading to the completion of the feedback circuit.

In this circuit FXR induces the expression of a transcriptional repressor SHP (small heterodimer partner) which in turn binds to LRH-1 (liver receptor homolog), which is required in CYP7A activation. Additionally, both LXR and FXR are implicated in the regulation of several other gene products involved in cholesterol absorption, metabolism and transport.

Thus, the identification of potent, selective, small molecule FXR agonists, partial agonists and antagonists would be powerful tools and would have many potential applications. For example, such compounds would facilitate the in vivo analysis of FXR physiology in vivo. In addition, such compounds, in conjunction with DNA arraying technology, might allow for the discovery of new gene products under the control of FXR. Further, FXR modulators might find potential utility in the treatment of cholestasis and other disease states associated with aberrant levels, flow and release of bile acids. Moreover, in the absence of a crystal structure of FXR, a thorough structure-activity relationship (SAR) study of ligands that modulate the activity of FXR would allow for the delineation of the structural requirements for ligand binding and might aid in the design of future ligands and potential therapeutics.

SUMMARY OF THE INVENTION

In accordance with the present invention, the identification of novel potent FXR activators is described. Initial screening of a 10,000-membered, diversity-orientated library of benzopyran containing small molecules for FXR activation utilizing a cell-based reporter assay led to the identification of several lead compounds owning low micromolar activity (EC₅₀'s=5-10 μM). These compounds were systematically modified employing parallel solution-phase synthesis and solid-phase synthesis to provide numerous compounds that potently activate FXR. Two derivatives of invention compounds, bearing stilbene or biaryl moieties, contain members that are the most potent FXR agonists reported to date in cell-based assays. These compounds are useful as chemical tools to further define the physiological role of FXR as well as therapeutic leads for the treatment of diseases linked to cholesterol, bile acids and their metabolism and homeostasis.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 summarizes the efficacy of the functional assay for the identification of FXR agonists, using the known FXR agonist, chenodeoxycholic acid (CDCA).

DETAILED DESCRITTION OF THE INVENTION

In accordance with the present invention, there are provided compounds having the structure:

wherein:

-   -   A is a C3 up to C8 branched chain alkyl or substituted alkyl         group, a C3 up to C7 cycloalkyl or substituted cycloalkyl, an         optionally substituted aryl or an optionally substituted         heteroaryl,     -   X is —C(O)— or —CH₂—,     -   R is methyl or ethyl,     -   R¹ is H, hydroxy, alkoxy, benzoyloxy, mesityloxy, or         —OCH₂C(O)OC₂H₅,     -   R² is H or R² can cooperate with R³ to form a benzopyran,         wherein the pyran ring has the structure:         wherein:     -   R⁶ is not present if the pyran ring is unsaturated, or, if         present, is selected from H, —OR, wherein R is alkyl or acyl, or         R⁶ can cooperate with R⁷ to form a cyclic acetal, a cyclic         ketal, or a cyclopropyl moiety, and     -   only one of R⁷ and R⁸ is present if the pyran ring is         unsaturated, or R⁷ and R⁸ are independently H, carboxyl, cyano,         hydroxy, alkoxy, thioalkyl, aryl, or R⁷ and R⁸ taken together         comprise a carbonyl oxygen or an oxime nitrogen, or either R⁷ or         R⁸ can cooperate with R⁶ to form a cyclic acetal, a cyclic         ketal, or a cyclopropyl moiety,

R³ can cooperate with R² to form a benzopyran having the structure set forth above, or R³ is alkenyl, optionally substituted aryl or heteroaryl, or optionally substituted arylalkenyl or heteroarylalkenyl,

R⁴ is H or hydroxy, and

R⁵ is H, hydroxy, alkoxy or aryloxy.

As employed herein, “alkyl” refers to saturated straight or branched chain hydrocarbon radical having in the range of 1 up to about 20 carbon atoms. “Lower alkyl” refers to alkyl groups having in the range of 1 up to about 5 carbon atoms. “Substituted alkyl” refers to alkyl groups further bearing one or more substituents selected from hydroxy, alkoxy (of a lower alkyl group), mercapto (of a lower alkyl group), cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, aryloxy, substituted aryloxy, halogen, trifluoromethyl, cyano, nitro, nitrone, amino, amido, (O)H, acyl, oxyacyl, carboxyl, carbamate, dithiocarbamoyl, sulfonyl, sulfonamide, sulfuryl, and the like.

As employed herein, “alkenyl” refers to straight or branched chain hydrocarbyl groups having at least one carbon-carbon double bond, and having in the range of about 2 up to 20 carbon atoms, and “substituted alkenyl” refers to alkenyl groups further bearing one or more substituents as set forth above.

As employed herein, “alkoxy” refers to —O-alkyl groups having in the range of 2 up to 20 carbon atoms and “substituted alkoxy” refers to alkoxy groups further bearing one or more substituents as set forth above.

As employed herein, “cycloalkyl” refers to a cyclic ring-containing groups containing in the range of about 3 up to about 8 carbon atoms, and “substituted cycloalkyl” refers to cycloalkyl groups further bearing one or more substituents as set forth above.

As employed herein, “heterocyclic” refers to cyclic (i.e., ring-containing) groups containing one or more heteroatoms (e.g., N, O, S, or the like) as part of the ring structure, and having in the range of 3 up to 14 carbon atoms and “substituted heterocyclic” refers to heterocyclic groups further bearing one or more substituents as set forth above.

As employed herein, “aryl” refers to aromatic groups having in the range of 6 up to 14 carbon atoms and “substituted aryl” refers to aryl groups further bearing one or more substituents as set forth above.

As employed herein, “aryloxy” refers to —O-aryl groups having in the range of 6 up to 14 carbon atoms and “substituted aryloxy” refers to aryloxy groups further bearing one or more substituents as set forth above.

As employed herein, “arylalkenyl” refers to aryl-substituted alkenyl groups and “substituted arylalkenyl” refers to arylalkenyl groups further bearing one or more substituents as set forth above.

As employed herein, “heteroaryl” refers to aromatic groups having in the range of 4 up to about 13 carbon atoms, and at least one heteroatom selected from O, N, S, or the like; and “substituted heteroaryl” refers to heteroaryl groups further bearing one or more substituents as set forth above.

As employed herein, “heteroarylalkenyl” refers to heteroaryl-substituted alkenyl groups and “substituted heteroarylalkenyl” refers to heteroarylalkenyl groups further bearing one or more substituents as set forth above.

As employed herein, “acyl” refers to alkyl-carbonyl species.

As employed herein, “halogen” refers to fluoride, chloride, bromide or iodide atoms.

As employed herein, reference to “a carbamate group” embraces substituents of the structure O—C(O)—NR₂, wherein each R is independently H, alkyl, substituted alkyl, aryl or substituted aryl as set forth above.

As employed herein, reference to “a dithiocarbamate group” embraces substituents of the structure —S—C(S)—NR₂, wherein each R is independently H, alkyl, substituted alky, aryl or substituted aryl as set forth above.

As employed herein, reference to “a sulfonamide group” embraces substituents of the structure —S(O)₂—NH₂.

As employed herein, “sulfeuryl” refers to substituents of the structure ═S(O)₂.

As employed herein, “amino” refers to the substituent —NH₂.

As employed herein, “monoalkylamino” refers to a substituent of the structure —NHR, wherein R is alkyl or substituted alkyl as set forth above.

As employed herein, “dialkylamino” refers to a substituent of the structure —NR₂, wherein each R is independently alkyl or substituted alkyl as set forth above.

As employed herein, reference to “an amide group” embraces substituents of the structure —C(O)—NR₂, wherein each R is independently H, alkyl, substituted alkyl, aryl or substituted aryl as set forth above. When each R is H, the substituent is also referred to as “carbamoyl” (i.e., a substituent having the structure —C(O)—NH₂). When only one of the R groups is H, the substituent is also referred to as “monoalkylcarbarnoyl” (i.e., a substituent having the structure —C(O)—NHR, wherein R is alkyl or substituted alkyl as set forth above) or “carylcarbamoyl” (i.e., a substituent having the structure —C(O)—NH(aryl), wherein aryl is as defined above, including substituted aryl). When neither of the R groups are H, the substituent is also referred to as “di-alkylcarbamoyl” (i.e., a substituent having the structure —C(O)—NR₂, wherein each R is independently alkyl or substituted alkyl as set forth above).

In accordance with a particular embodiment of the present invention, presently preferred compounds are those wherein A is a C5-C7 cycloalkyl group.

In accordance with another particular embodiment of the present invention, presently preferred compounds are those wherein X is —C(O)—.

In accordance with yet another particular embodiment of the present invention, presently preferred compounds are those wherein R¹ is hydrogen.

In accordance with still another particular embodiment of the present invention, presently preferred compounds are those wherein R² and R³ cooperate to form a benzopyran.

In accordance with a further particular embodiment of the present invention, presently preferred compounds are those wherein R³ is alkenyl, thereby producing a cinnamate derivative.

In accordance with a still further embodiment of the present invention, presently preferred compounds are those wherein R³ is an optionally substituted aryl or heteroaryl moiety, thereby producing biphenyl derivatives.

In accordance with yet another embodiment of the present invention, presently preferred compounds are those wherein R³ is an optionally substituted arylalkenyl or heteroarylalkenyl moiety, thereby producing stilbene derivatives.

As there was, prior to the present invention, only one example of a high affinity, non-steroidal agonist for FXR, i.e., GW 4064 (3, having an EC₅₀=80 nM, structure shown below), the strategy adopted herein for identification of additional potent compounds involved screening a 10,000-membered library constructed around the privileged 2,2-dimethylbenzopyran scaffold.

Such privileged structures are attractive starting points for lead compound discovery, particularly when there exists little structural information regarding the target, as they show good binding affinity toward a wide variety of enzymes and receptors. The initial hits discovered from screening of this library for FXR activation could be further modified for enhanced potency and pharmacological properties suitable for the applications mentioned above. Implementation of such a strategy is described herein, culminating in the discovery of numerous potent and selective activators of FXR.

Thus, in accordance with the present invention, a cell-based transcription assay is employed in which an FXR responsive promoter is linked to a luciferase reporter as the primary screen (see Example 1). In addition to ensuring that only cell permeable compounds were selected for further optimization, this approach allows for the detection of FXR activation in a natural system (i.e., correct folding of the protein and in the presence of a complete compliment of co-activators and co-repressors). Initial screening of a 10,000-membered combinatorial library of benzopyran-based small molecules in this high-throughput, cell-based assay for FXR activation produced several lead compounds, 4-15, whose structures are shown below:

Guided by the preliminary structure-activity relationships (SAR) gained from the evaluation of this initial library, a follow-up focused library of about 200 benzopyran-based compounds was designed and synthesized on solid support employing the protocol set forth below. Library construction

Scaltoids

Electrophiles

Amines

A selection of the most active compounds, possessing activities from 5-10 μM, discovered from this second round of screening, includes compounds 16-27, as shown below:

Compounds 26 and 27 proved to be among the most active at this stage and were the subject of further modification as described below.

With initial lead compounds identified and validated, the stage is set for the systematic modification of the three regions of lead compound 26, as shown below:

As detailed in the following sections, focused libraries were synthesized and screened in the cell-based assay in order to evaluate the structural requirements of each region of the molecule for potent FXR agonism. At this point parallel solution-phase chemistry was selected for the construction of additional focused libraries. This shift away from solid-phase chemistry provided maximum flexibility in efforts to rapidly and systematically modify each region of the lead molecules using smaller designed libraries.

Most of the FXR agonists reported to date, including CDCA (1; see structure above), TINPB (2; structure shown below) and GW4064 (3; see structure above), contain a carboxylic acid moiety.

It was reasoned, therefore, that incorporation of an acid unit within either region I, II or III of lead compound 26 (as illustrated above) would confer increased potency upon this rather weak ligand (5-10 μM) identified via HTS. Evaluation of the Benzopyran Region I SAR

Guided by this reasoning, the SAR of region I was evaluated. Several compounds displaying the acid unit in various positions (e.g., Compounds 28, 36, 52, 54 and 56), were prepared (see, Examples 3 to 6) and tested.

R =

None of these compounds, however, showed improved activation of FXR. Interestingly, compound 29, bearing a meta methyl acrylate moiety, was a substantially better activator of FXR than compound 26.

In further refining the SAR of region I, it was observed that the location of the methyl acrylate moiety at the meta position was beneficial to achieve potent activation of FXR, as compound 53, bearing a para methyl acrylate, does not activate FXR under the conditions tested. In order to further examine what functionality was tolerable at the meta position, additional compounds with meta substituents (as shown above) were synthesized. From biological screening of these compounds it became clear that the length and rigidity of the tether between the aromatic core and the interacting functionality (either methyl ester or methyl ether) are important for FXR agonism. For instance, compounds 41 and 45 appear to possess either too short or too long of a tether for potent activity; compounds 35 and 46-49 presumably cannot adopt the correct orientation for potent activation; and compounds 30, 31, 34, 38, 39, 40 and 50 do not apparently present the correct interacting functionality to the receptor as they are inactive. Indeed, of all the analogs designed to probe the SAR of region I, only compounds 29 and 33 are capable of activating FXR to a significant extent. Due to relative ease of synthesis of compound 29 this analog was chosen as a starting point for the modification of region II.

Evaluation of the Benzopyran Region II SAR

Benzopyran region II SAR was evaluated through traditional solution phase chemistry to see the effect of various substitution patterns in this region of the molecule. Thus, compounds 61-84 (structures shown below) were prepared (see, Example 7) and tested.

R =

Only compounds 65 (EC₅₀=358 nM) and 68 EC₅₀=1,000 nM) were more effective than compound 29 in activating FXR. Substituted aromatic amide derivatives such as 69-77 were all found to be less active than the parent compound 68. Alkyl derivatives 78 and 79 were inactive as were sulfonamide 82, thiourea 84, and thioamide 83, suggesting the importance of acylation at this position. The sum of these results pointed to the desirable presence of moderately bulky cycloalkyl amide moieties in region II for good activity.

Evaluation of the Benzopyran Region III SAR

Having thoroughly examined regions I and II, the modification of region III was then undertaken. Thus, compounds 85-102 (structures shown below, along with compound 68 for ease of comparison) were prepared (see Examples 8 and 9) and tested.

R¹, R² =

Incorporation of polar H-bond donating functional groups such as those that adorn compounds 86, 93, 94, 98 and 100 did not improve the activity of the analogs. Nor did the addition of H-bond acceptors such as in 89, 90, 95, 99 and 101 improve the ability of the parent compound 68 to activate FXR.

The addition of bulky lipophilic groups to the benzopyran moiety afforded compounds that only weakly activated FXR. However, replacement of the double bond in the benzopyran unit by a dichlorocyclopropane unit provided analog 102 (EC₅₀=333 nM). Replacement of the benzoyl group in region II of compound 102 with the cyclohexylcarbonyl moiety afforded the even more potent compound 149 (EC₅₀=188 nM).

Although compound 149 (EC₅₀=188 nM) represents a significant improvement in potency over compound 65 (EC₅₀=348 nM), it was not readily apparent how the activity of this class of compounds could be further improved. Therefore, it was decided to examine the effect of replacing the benzopyran moiety with other ring systems.

Thus, a series of compounds (i.e., Compounds 104-129) in which the benzopyran moiety was replaced with certain groups of varying molecular diversity was prepared (see Examples 10 to 17) and tested.

R =

Biological assays showed that replacement of the benzopyran with a small aromatic unit generally had a detrimental effect on activity. For instance, compounds 110 and 112-117 were inactive, while compounds 111 and 118 showed only moderate activation of FXR (EC₅₀=680 nM and 606 nM, respectively; see Table 3 in Example 1). However, replacement of the benzopyran with an aromatic ring bearing substituents at the para position produced compounds with improved activity. For example, 4-tert-butyl cinnamate 105 (EC₅₀=127 nM), stilbenes 121 and 122 (EC₅₀=36 and 208 nM, respectively), biaryls 124-127 (EC₅₀=510, 69, 77, 227 nM, respectively) and aryl thiophenes 128 and 129 (EC₅₀=206 and 256 nM, respectively) were all potent activators of FXR in the cell-based reporter assay (see Tables 1, 9, 10 and 11 in Example 1).

This initial survey of the three regions of SAR outlined above led to the identification of several potent FXR agonists for further evaluation. One such agonist is the benzopyran-derived dichlorocyclopropane 149 (EC₅₀=188 nM). Compound 105 (EC₅₀=127 nM) is an example of a bis-cinnamate derivative. Finally, compounds 121 (EC₅₀=36 nM) and 124 (EC₅₀=69 nM) are stilbene and biaryl derivatives, respectively, of invention compounds.

Based on the results observed thus far, compound 149 appeared to represent the most potent derivative that could be readily obtained among the benzopyran-derivatives. However, the bis-cinnamate, biaryl, and stilbene derivatives of invention compounds were thought to still possess considerable potential for further development and rigorous SAR analysis. Below the results of such investigations are detailed, which indeed led to further enhancement of biological activity.

Examination of the Bis-Cinnamate Series

Similar to the results described above, the meta substituted methyl cinnamate moiety on the “right-hand” region of the molecule remained a desirable component for elevated activity among the bis-cinnamate derivatives of invention compounds (see compounds 105 and 133-139).

Replacement of the methyl acrylate unit with either a methyl or ethyl allylic ether (compounds 136 and 137) caused only a slight decrease in activity (EC₅₀=243 and 220 nM, respectively). A marked decline in potency accompanied substitution of the methyl acrylate by more sterically bulky ethers or esters (compounds 133 and 134) or amides (compound 135). Interestingly, saturation of the acrylate olefin (compound 139) afforded only a two-fold decrease in potency, EC₅₀=274 nM, which supports the notion that conformational rigidity is a factor contributing to, but not essential for, high affinity ligands. Importantly, compound 139 suggests that the methyl acrylate moiety is not simply functioning as a latent electrophile.

Region II also closely mirrored the preceding data as cycloalkyl amides remained the preferred substituents (see, compounds 105 and 140-145: EC₅₀=127-250 nM) among the bis-cinnamate derivatives of invention compounds. Aromatic and heterocyclic amides as well as alkyl ureas led to moderate potency (compounds 143-145: EC₅₀=205-236 nM) whereas incorporation of bulky ureas such as compound 146 rendered compounds of only marginal efficacy.

As mentioned above, replacement of the benzopyran moiety with a benzyl group bearing a tert-butyl acrylate moiety in the para-position yielded compound 105 with dramatically increased efficacy (EC₅₀=127 nM). Interestingly, placement of the same tert-butyl acrylate group in either the meta or ortho positions of the aromatic ring in Region III led to only micromolar potency (see compounds 107 and 109). Further investigation of the “left-hand” region in this series of compounds demonstrated that a decrease in ester group size yielded a corresponding decrease in efficacy (EC₅₀ of t-butyl>i-propyl>ethyl>methyl (see, compounds 105 and 150-152). Similarly, substitution of the ester with either carboxylic acid or amide functionality provided less effective compounds with EC₅₀ values in the micromolar range. Substitution of the tert-butyl acrylate moiety with a methyl or ethyl allylic ether (see, compounds 156 and 157) retained considerable potency (EC₅₀=233 and 198 nM, respectively). However, the more bulky phenyl allylic ether 158 possessed only micromolar activity. In addition, saturation of the acrylate moiety (compound 159) showed a two-fold decrease in potency from the parent compound 105. Finally, substitution of the ortho position of the aromatic ring of the tert-butyl acrylate series with oxygenated functionality afforded compounds with very low biological activity (see compounds 161-167).

Construction of Biaryl and Stilbene Containing Focused Libraries

In an effort to further explore the activities of biaryl and stilbene derivatives of invention compounds, a 94-membered library of such compounds was constructed employing a solid phase strategy (see Example 18).

The selection of appropriate styrenes and boronic acids for inputs into this combinatorial library was guided by initial comparisons of tert-butyl stilbene (compound 123, EC₅₀=>1000 nM) to the unsubstituted stilbene 102 (EC₅₀=36 DM), and biaryl compound 124 (EC₅₀=510 nM) to compound 125 (EC₅₀=69 nM). It was reasoned that both the stilbene and the biaryl ligands needed to fit into the same region of space within the receptor site for potent activation. Thus, stilbenes in which the aromatic nucleus is removed two carbon atoms further away from the core of the molecule should be adorned with small substituents while the biaryl compounds should be adorned with larger functionality for optimal activity. Screening of this compound library in the cell-based assay led to some intriguing results as summarized in Tables 12-15 of Example 1.

Thus, it was found that in both stilbene and biaryl derivatives of invention compounds, analogs bearing the cyclohexylamide moiety are generally the most potent followed by those bearing the isopropyl amide or isopropyl urea units. As predicted above, stilbenes bearing smaller substituents were more potent than those bearing larger functionality. For instance, substituted stilbene 121 and mono-fluoro stilbenes 192, 201, and 204 were among the most active, while mono-methyl derivative 174 and tri-methyl derivative 195 were among the least active. Also of interest were heterocyclic compounds 207 and 210, which retained good potency (EC₅₀=309 and 227 nM, respectively) and may possess improved pharmacological properties. With biaryl derivatives of invention compounds, compounds which present more bulk at the terminus of the structure were more active. With these derivatives, compounds 259 (EC₅₀=25 nM) and 244 (EC₅₀=38 nM) were particularly active. Overall, most of the compounds synthesized in this follow-up library were efficient activators of FXR, confirming the accuracy of the working hypothesis for the FXR binding pocket described above, which provides a solid basis for further development of FXR activators.

A summary of the molecular requirements for potent FXR activation is shown below:

Thus, in Region I the presence of the meta methyl acrylate unit or allylic methyl ether is desirable for potent activation as only a few modifications retain good activity. In some instances, when other areas are modified, the olefin can be deleted from Region I while retaining potency. The most potent compounds observed thus far possess an amide or a urea in Region II. Presently most preferred compounds have a cycloalkylamide or a cycloalkylurea in Region II. Finally, Region III is the most tolerant, indeed, several structural elements were found to provide a good fit within the pocket of the receptor. Generally, it is desirable for the aromatic ring to be para-substituted, with the steric bulk and length of the substituent imparting a significant effect on potency of the resulting compound.

In order to determine how selectively the above-described compounds activated FXR, some of the most active compounds were screened against a panel of nuclear receptors. Most of these compounds were found to be selective for activation only of FXR. Notably, however, compound 149 also potently activated SXR. This result may lead to compounds which have utility in the treatment of diseases linked to the accumulation of toxic bile acids.

In accordance with another embodiment of the present invention, there are provided formulations comprising at least one of the above-described compounds in a pharmaceutically acceptable carrier therefor. Exemplary pharmaceutically acceptable carriers include solids, solutions, emulsions, dispersions, micelles, liposomes, and the like. Optionally, the pharmaceutically acceptable carrier employed herein further comprises an enteric coating.

Pharmaceutically acceptable carriers contemplated for use in the practice of the present invention are those which render invention compounds amenable to oral delivery, transdermal delivery, intravenous delivery, intramuscular delivery, topical delivery, nasal delivery, and the like.

Thus, formulations of the present invention can be used in the form of a solid, a solution, an emulsion, a dispersion, a micelle, a liposome, and the like, wherein the resulting formulation contains one or more of the compounds of the present invention, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for enterable or parenteral applications. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions and any other suitable for use. The carriers which can be used include glucose, lactose, gum acacia, gelatin, manitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, medium chain length triglycerides, dextrans, and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form. In addition auxiliary, stabilizing, thickening, and coloring agents and perfumes may be used. The active compound(s) is (are) included in the formulation in an amount sufficient to produce the desired effect upon the process or disease condition.

Invention formulations containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Formulations intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such formulations may contain one or more agents selected from the group consisting of a sweetening agent such as sucrose, lactose, or saccharin, flavoring agents such as peppermint, oil of wintergreen or cherry, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients used may be, for example (1) inert diluents such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; (2) granulating and disintegrating agents such corn starch, potato starch or alginic acid; (3) binding agents such as gum tragacanth, corn starch, gelatin or acacia, and (4) lubricating agents such as magnesium stearate, steric acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by such techniques as those described in U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874, to form osmotic therapeutic tablets for controlled release.

In some cases, formulations contemplated for oral use may be in the form of hard gelatin capsules wherein the active ingredient is mixed with inert solid diluent(s), for example, calcium carbonate, calcium phosphate or kaolin. They may also be in the form of soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.

Invention formulations may be in the form of a sterile injectable suspension. This suspension may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides, fatty acids, naturally occurring vegetable oils like sesame oil, coconut oil, peanut oil, cottonseed oil, etc. or synthetic fatty vehicles like ethyl oleate or the like. Buffers, preservatives, antioxidants, and the like can be incorporated as required.

Invention formulations may also be administered in the form of suppositories for rectal administration of the drug. These formulations may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters of polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug. Since individual subjects may present a wide variation in severity of symptoms and each drug has its unique therapeutic characteristics, the precise mode of administration and dosage employed for each subject is left to the discretion of the practitioner.

Amounts effective for the particular therapeutic goal sought will, of course, depend on the severity of the condition being treated, and the weight and general state of the subject. Various general considerations taken into account in determining the “effective amount” are known to those of skill in the art and are described, e.g., in Gilman et al., eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990, each of which is herein incorporated by reference.

The term “effective amount” as applied to invention compounds, means the quantity necessary to effect the desired therapeutic result, for example, a level effective to treat, cure, or alleviate the symptoms of a disease state for which the therapeutic compound is being administered, or to establish homeostasis. Since individual subjects may present a wide variation in severity of symptoms and each drug or active agent has its unique therapeutic characteristics, the precise mode of administration, dosage employed and treatment protocol for each subject is left to the discretion of the practitioner.

In accordance with yet another embodiment of the present invention, there are provided methods for modulating process(es) mediated by farnesoid X receptor polypeptides, said methods comprising conducting said process(es) in the presence of an effective amount of at least one compound according to the invention.

As employed herein, “modulating” refers to the ability of a modulator for a member of the nuclear receptor superfamily (e.g., FXR) to either directly (by binding to the receptor as a ligand) or indirectly (as a precursor for a ligand or an inducer which promotes production of ligand from a precursor) induce expression of gene(s) maintained under hormone expression control, or to repress expression of gene(s) maintained under such control. Exemplary processes contemplated for modulation according to the invention include cholesterol metabolism, regulation of lipid homeostasis, stimulation of bile transport and absorption, regulation of the expression of genes involved in the excretion and transportation of bile acids (including intestinal bile acid-binding protein (IBABP)), bile salt export pump (BSEP) and canalicular multi-specific organic anion transporter (cMOAT), and the like.

Bile acids are derivatives of cholesterol synthesized in the hepatocyte. Cholesterol, ingested as part of the diet or derived from hepatic synthesis is converted into the bile acids cholic and chenodeoxycholic acids, which are then conjugated to an amino acid (glycine or taurine) to yield the conjugated form that is actively secreted into cannaliculi. Bile acids are facial amphipathic, that is, they contain both hydrophobic (lipid soluble) and polar (hydrophilic) faces. The cholesterol-derived portion of a bile acid has one face that is hydrophobic (that with methyl groups) and one that is hydrophilic (that with the hydroxyl groups); the amino acid conjugate is polar and hydrophilic. Therefore, compounds that can be used to modulate such pathways via effects on FXR involving bile acids are useful in cholesterol metabolism.

Bile acid synthesis is a major pathway for cholesterol disposal and thus represents a potential therapeutic target pathway for the treatment of hypercholesterolemia. FXR acts as a bile acid receptor and biological sensor for the regulation of bile acid biosynthesis. FXR is known to regulate cholesterol metabolism in two ways: (1) chenodeoxycholic acid (CDCA), a primary bile acid, binds directly to and activates FXR, which then mediates the feedback suppression by bile acids of cholesterol 7 alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid biosynthesis from cholesterol; and (2) FXR participates in the activation of intestinal bile acid binding protein (IBABP), which is involved in the enterohepatic circulation of bile acids. Thus FXR constitutes a potential therapeutic target that can be modulated to enhance the removal of cholesterol from the body. Novel compounds identified by the methods presented herein provide a new tool for regulating or modulating FXR function.

Furthermore, FXR is known to in turn activate a series of target genes. In particular FXR functions as a heterodimer with the 9-cis-retinoic acid receptor (RXR). A number of target DNA binding sequences that would be present in target genes have recently been identified. A consensus sequence has been determined, which contains an inverted repeat of the sequence AGGTCA with a 1-base pair spacing (IR-1) (Laffitte et al., J. Biol. Chem. 275:10638-10647 (2000). This sequence was shown to be a high affinity binding site for FXR/RXR in vitro and to confer ligand-dependent transcriptional activation by FXR/RXR to a heterologous promoter in response to a bile acid or synthetic retinoid. Although these studies demonstrated that the FXR/RXR heterodimer binds to the consensus IR-1 sequence with the highest affinity, it was also demonstrated that FXR/RXR can bind to and activate through a variety of elements including IR-1 elements with changes in the core half-site sequence, spacing nucleotide, and flanking nucleotides. In addition, it was shown that FXR/RXR can bind to and transactivate through direct repeats. Therefore, by providing novel ways to modulate FXR function, the present invention in turn provides a method of modulating the function of a variety of target genes that are acted upon by FXR.

In accordance with still another embodiment of the present invention, there are provided methods for the treatment of hypercholestemia, said methods comprising administering an effective amount of at least one compound according to the invention to a subject in need thereof.

In accordance with still another embodiment of the present invention, there are provided methods for the treatment of cholestasis, said methods comprising administering an effective amount of at least one compound according to the invention to a subject in need thereof.

The invention will now be described in greater detail with reference to the following non-limiting Examples.

EXAMPLE 1 In Vivo Assay

The feasibility of creating high throughput screens (XIS) for ORs was explored using FXR as a candidate orphan receptor (OR) with a known activator, chenodeoxycholic acid (CDCA) as a ligand. The screen is based on the co-transfection of a full-length receptor with the reporter vector containing a natural hormone response element under a minimal eukaryotic promoter. The results provided herein (see, FIG. 1) demonstrate that compounds can be successfully screened in a dose dependent manner for potential activating chemical ligands using a full length FXR on a natural response element. These results validate the robustness of the assay for FXR, in 384-well plates. Using this 384-well format, the high throughput screening (HTS) approach to FXR as a candidate OR was employed. For this test screen, a 10,000 membered library, constructed around the privileged 2,2-dimethylbenzopyran scaffold, was employed (see Nicolaou et al., J. Am. Chem. Soc. 122:9939-9953 and 9954-9967 (2000)). This library comprises approximately 10,000 distinct compounds with structures and sizes similar to natural products such as phyto-estrogens, flavanoids, coumarins and long chain fatty acids. A central question in the feasibility studies is whether this library is suitable for screening for nuclear receptor ligands. Samples of this library were first reformatted into a 384-well format and then subjected to the FXR cell-based assays described above and assessed for FXR-mediated transcriptional activity. Cells were exposed to approximately 10 μM of sample for 18 hrs prior to washing and luciferase analysis.

The 25 most active compounds at 10 μM were re-synthesized to confirm their structure and activity. Smaller “focused” chemical libraries were then designed and prepared around these hits and subjected to multiple rounds of screening. Through this iterative process a total of seven additional rounds of synthesis and selection was conducted resulting in novel compounds that are as effective as a proprietary synthetic ligand developed by Glaxo-Smith-Kline (GW 4064) in cell based assays. Using one of these identified compounds, fexaramate (105; EC₅₀ 127 nM), as a scaffold, three additional focused libraries were made and screened to obtain at least four potent, non-steroidal FXR agonists termed fexarene (121; EC₅₀, 36 nM), fexaramine (259; EC₅₀, 25 nM), fexarine (244; EC₅₀, 38 nM) and fexarchloramide (149; EC₅₀, 188 nM). EC₅₀ values were determined with Prism 3.0 software via the activity of the subject compound in the previously described cell based assay.

EC₅₀ values for the “scaffold” compound, fexaramate (105), and numerous variations thereof, are presented below (see Tables 1-11). TABLE 1

R EC₅₀ (nM) RE^(a) 105 COOMe 127 2.12 123 COOEt 256 2.07 134 COO^(t)Bu >1000 1.06 135 CONH₂ >1000 0.50 136 CH₂OMe 243 1.88 137 CH₂OEt 220 1.74 138 CH₂OPh 2830 0.45 139: EC₅₀ = 274 nM RE^(a) = 1.38 ^(a)SAR of region I

TABLE 2

R EC₅₀ (nM) RE^(b) 140 cyclopropyl 250 1.88 141 cyclobutyl 187 1.84 142 cyclopentyl 162 2.15 105 cyclohexyl 127 2.12 143 phenyl 238 1.96 144 2-furyl 205 1.93 145 isopropylamino 212 1.96 146 benzylamino >1000 0.27 ^(b)SAR of region II

TABLE 3

R EC₅₀ (nM) RE^(c) 132 H >1000 0.09 147 methyl >1000 0.09 110 benzyl >1000 0.09 111 2-naphthyl 680 0.41 114 2-bromobenzyl >1000 0.11 113 3-bromobenzyl >1000 0.10 112 4-bromobenzyl >1000 0.28 148 4-tert-butylbenzyl >1000 0.15 115 3-methoxybenzyl >1000 0.11 116 3,5-dimethoxybenzyl 606 0.11 117 3-(trifluoromethyl)benzyl >1000 0.12 ^(c)SAR of region III

TABLE 4

R EC₅₀ (nM) RE^(a) 68 phenyl >1000 0.83 65 cyclohexyl 358 0.40

TABLE 5

R EC₅₀ (nM) RE^(b) 102 phenyl 333 0.64 149 cyclohexyl 188 0.50

TABLE 6

R EC₅₀ (nM) RE^(d) 104 COOH >1000 0.08 150 COOMe >1000 0.87 151 COOEt >1000 1.14 152 COOPr 163 1.97 105 COO^(i)Bu 127 2.12 153 COOBn >1000 0.23 154 CONMe₂ >1000 0.66 155 CONH^(i)Bu >1000 1.85 156 CH₂OMe 233 1.63 157 CH₂OEt 198 2.06 158 CH₂OPh >1000 0.64 ^(d)SAR of region III

TABLE 7

R EC₅₀ (nM) RE^(b) 159 COOMe 240 1.56 160 COO^(i)Bu >1000 0.84

TABLE 8

R EC₅₀ (nM) RE^(a) 161 H >1000 0.12 162 Me >1000 0.14 163 Bn >1000 0.38 164 MeC(O) >1000 0.16 165 C₆H₅C(O) >1000 0.16 166 MeS(O₂) >1000 0.16 167 EtOOCCH₂ >1000 0.16

TABLE 9

R¹ R² EC₅₀ (nM) RE^(a) i OMe H 77 1.51 127 C(O)Me H 227 1.30 125 H SMe 89 1.74 124 H H 510 0.71

TABLE 10

R EC₅₀ (nM) RE^(b) 128 Me 206 1.78 129 C(O)Me 256 1.48

TABLE 11

R EC₅₀ (nM) RE^(a) 121 H 36 1.55 122 OMe 208 1.67 123 i-Bu >1000 0.29

Several of the derivatives set forth above are seen to possess excellent activity (e.g., Compounds 121, 125, 141, 142, etc.).

EC₅₀ values for numerous additional variations of the compounds presented above are presented below (see Tables 12-15). TABLE 12

R¹ R² R³ R⁴ R⁵ R⁶ EC₅₀ (nM) RE^(a) 174 H H Me H H —C₅H₁₁ 342 0.83 175 H H Me H H —CH(CH₃)₂ 1410 0.37 176 H H Me H H —NHCH(CH₃)₂ 3570 0.10 177 Cl H H H Cl —C₅H₁₁ 150 0.12 178 Cl H H H Cl —CH(CH₃)₂ 195 0.14 179 Cl H H H Cl —NHCH(CH₃)₂ 216 0.15 180 H Cl H H H —C₅H₁₁ 165 1.41 181 H Cl H H H —CH(CH₃)₂ 264 1.09 182 H Cl H H H —NHCH(CH₃)₂ 339 0.58 153 H CF₃ H CF₃ H —C₅H₁₁ 1470 0.1S 184 H CF₃ H CF₃ H —CH(CH₃)₂ 1950 0.13 185 H CF₃ H CF₃ H —NHCH(CH₃)₂ 1830 0.13 188 H CF₃ H H H —C₅H₁₁ 937 0.35 187 H CF₃ H H H —CH(CH₃)₂ 267 0.70 188 H CF₃ H H H —NHCH(CH₃)₂ 932 0.31 189 F H H H F —C₅H₁₁ 174 0.94 190 F H H H F —CH(CH₃)₂ 108 0.79 191 F H H H F —NHCH(CH₃)₂ 4020 0.21 192 F H H H H —C₅H₁₁ 64 1.41 193 F H H H H —CH(CH₃)₂ 70 1.17 194 F H H H H —NHCH(CH₃)₂ 431 0.89 195 Me H Me H Me —C₅H₁₁ 518 0.24 196 Me H Me H Me —CH(CH₃)₂ 149 0.30 197 Me H Me H Me —NHCH(CH₃)₂ 431 0.14 198 H H H H H —CH(CH₃)₂ 65 1.33 200 H H H H H —NHCH(CH₃)₂ 119 1.38 201 H F H H H —C₅H₁₁ 86 1.38 202 H F H H H —CH(CH₃)₂ 71 1.33 203 H F H H H —NHCH(CH₃)₂ 467 0.61 204 H H F H H —C₅H₁₁ 185 0.53 205 H H F H H —CH(CH₃)₂ 120 1.19 206 H H F H H —NHCH(CH₃)₂ 348 0.91

TABLE 13

R EC₅₀ (nM) RE^(b) 207 —C₅H₁₁ 309 0.61 208 —CH(CH₃)₂ 310 0.62 209 —NHCH(CH₃)₂ 575 0.68

TABLE 14

R EC₅₀ (nM) RE^(b) 210 —C₅H₁₁ 227 0.53 211 —CH(CH₃)₂ 228 0.32 212 —NHCH(CH₃)₂ 356 0.42

TABLE 15

R¹ R² R³ R⁴ R⁵ R⁶ EC₅₀ (nM) RE^(a) 213 H F F H H —C₅H₁₁ 72 1.70 214 H F F H H —CH(CH₃)₃ 249 1.15 215 H F P H H —NHCH(CH₃)₂ 8180 0.23 125 H H SMe H H —C₅H₁₁ 69 1.74 216 H H SMe H H —CH(CH₃)₃ 51 0.98 217 H H SMe H H —NHCH(CH₃)₂ 178 0.23 218 OMe H H H H —C₅H₁₁ 399 0.40 219 OMe H H H H —CH(CH₃)₂ 377 0.28 220 OMe H H H H —NHCH(CH₃)₂ 4010 0.09 126 H Cl H Cl H —C₅H₁₁ 284 0.95 221 H Cl H Cl H —CH(CH₃)₃ 561 0.54 222 H Cl H Cl H —NHCH(CH₃)₂ >10000 0.10 223 H OMe H H H —C₅H₁₁ 101 1.51 224 H OMe H H H —CH(CH₃)₂ 72 128 225 H OMe H H H —NHCH(CH₃)₂ 1370 0.41 226 H OEt H H H —C₅H₁₁ 147 1.37 227 H OEt H H H —CH(CH₃)₂ 173 1.03 228 H OEt H H H —NHCH(CH₃)₂ 2350 0.33 229 H H OMe H H —C₅H₁₁ 89 1.71 230 H H OMe H H —CH(CH₃)₂ 97 121 231 H H OMe H H —NHCH(CH₃)₂ 144 1.16 232 H Cl H H H —C₅H₁₁ 94 1.56 223 H Cl H H H —CH(CH₃)₂ 77 1.52 234 H Cl H H H —NHCH(CH₃)₂ 1400 0.49 235 H H Me H H —C₅H₁₁ 26 1.38 236 H H Me H H —CH(CH₃)₂ 118 1.48 237 H H Me H H —NHCH(CH₃)₂ 449 0.80 238 H Me H H H —C₅H₁₁ 109 1.43 239 H Me H H H —CH(CH₃)₂ 163 1.09 240 H Me H H H —NHCH(CH₃)₂ 1330 0.53 241 OMe H H Cl H —C₅H₁₁ 233 1.16 242 OMe H H Cl H —CH(CH₃)₂ 226 0.79 243 OMe H H Cl H —NHCH(CH₃)₂ 3080 0.17 244 H —OCH₂O— H H —C₅H₁₁ 38 1.90 245 H —OCH₂O— H H —CH(CH₃)₂ 19 1.25 246 H —OCH₂O— H H —NHCH(CH₃)₂ 96 1.51 247 H Cl F H H —C₅H₁₁ 66 1.67 248 H Cl F H H —CH(CH₃)₂ 129 1.64 249 H Cl F H H —NHCH(CH₃)₂ 3050 0.41 250 H H OCF₃ H H —C₅H₁₁ 264 1.04 251 H H OCF₃ H H —CH(CH₃)₂ 219 0.78 252 H H OCF₃ H H —NHCH(CH₃)₂ 7530 0.21 253 H OCF₃ H H H —C₅H₁₁ 420 0.84 254 H OCF₃ H H H —CH(CH₃)₂ 247 0.59 255 H OCF₃ H H H —NHCH(CH₃)₂ >10000 0.09 256 OMe H H H OMe —C₅H₁₁ 77 0.12 257 OMe H H H OMe —CH(CH₃)₂ 95 0.10 258 OMe H H H OMe —NHCH(CH₃)₂ 561 0.10 259 H H NMe₂ H H —C₅H₁₁ 25 1.72 260 H H NMe₂ H H —CH(CH₃)₂ 57 1.07 261 H H NMe₂ H H —NHCH(CH₃)₂ 162 1.01 262 H H i-Bu H H —C₅H₁₁ 132 1.38 263 H H i-Bu H H —CH(CH₃)₂ 343 0.59 284 H H i-Bu H H —NHCH(CH₃)₂ 262 1.02

Several of the derivatives set forth above are seen to possess excellent activity (e.g., Compounds 177, 180, 181, 189, 190, 192, 193, 198, 201, 202, 204, 205, 213, 216, 224, 229, 230, 232, 233, 235, 236, 238, 244, 245, 246, 247, 256, 257, 259, 260, etc.).

EXAMPLE 2 In Vitro Screening

An in vitro based “proximity” screen is an excellent complement to live cell assays and can be used as a measure of direct ligand binding. Hence this type of screen is also an effective measure of the affinity of binding without the use of a radiolabel. The approach employed herein is termed AlphaScreen technology. For this assay purified receptor protein is expressed as a glutathione S-transferase (GST) fusion protein and is bound via a GST antibody to a “donor” bead. This is then mixed with a biotinylated co-activator peptide that has been linked to an Avidin proximity sensitive “acceptor” bead. These reactants are mixed in a 384-well plate and are then exposed to either a known inducer (control) or an ordered array of unknown compounds (library). If the acceptor bead (linked to the co-activator peptide) is brought into close proximity of the donor bead, by virtue of a biological interaction, singlet-state oxygen molecules are released and react with chemiluminescent groups in the acceptor beads. The effect of either known inducers or candidate chemical compounds on the interaction of a receptor and its co-activator peptide can be measured by a change in the Alpha signal.

The ability of the in vitro AlphaQuest assay to detect receptor/co-activator peptide interactions in a 384 well format has been evaluated using the thyroid hormone receptor (TR) and the retinoid X receptor (RXR) as positive controls. The results demonstrate that receptor/co-activator peptide interactions can be detected in a dose-dependent manner with binding efficiencies similar to those reported in the literature, validating this as a critical in vitro approach to demonstrate binding of candidate ligands in the absence of a high affinity radiolabeled competitor.

Representative procedures for the preparation of Region I modified compounds are shown in Examples 3 to 6.

EXAMPLE 3 Preparation of Compounds 29, 60, S-5, S-6 and S-9

The strategy employed for the preparation of Compound 29 is shown below.

Thus, 2,3-dihydroxy benzaldehyde 59 is selectively methylated (NaH, MeI) to afford the monoalcohol benzaldehyde S-5. S-5 is O-alkylated (1.5 equiv. of 2-methyl-3-butyn-2-ol, 1.7 equiv. of TFAA, 1.5 equiv. of DBU, 0.1 equiv. of CuCl₂, CH₃CN, 0-25° C., 12 h., 75%) to provide the propargyl ether S-6. S-6 is thermally cyclized (N,N-diethylaniline, 190° C., 0.5 h., 90%) to afford the intermediate benzopyran 60. 60 is reductively aminated (1.5 equiv. of 3-bromoaniline, THF, 70° C., 4 h., 90%, then 2.0 equiv. of NaCNBH₃, 10% MeOH, 70° C., 4 h., 83%) and the intermediate amine is acylated (1.3 equiv. of cyclopropanecarbonyl chloride (C₃H₅COCl), 1.3 equiv. of Et₃N, 0.1 equiv. of 4-DMAP, CH₂Cl₂, 25° C., 12 h., 85-95%) to provide the aryl bromide amide S-9. S-9 is coupled to methyl acrylate by a palladium-mediated Heck reaction (4.0 equiv. of methyl acrylate, 0.2 equiv. of Pd₂(dba)₃, 0.5 equiv. of P(o-tol)₃, 5.0 equiv. of Et₃N, DMF, 24 h., 80%) to afford compound 29.

EXAMPLE 4 Preparation of Compounds 28, 29, 36, 42, 51-56, S-7 and S-8

The strategy employed for the preparation of Compounds 28, 29, 36, 42, 51-56, S-7 and S-8 is shown below.

Thus, benzopyran aldehyde 60 is reductively aminated (1.5 equiv. of methyl 4-amino-benzoate, or ethyl 3-aminobenzoate, or methyl (4-aminomethyl)benzoate, THF, 70° C., 4 h., 70° C., then 2.0 equiv. of NaCNBH₃, 10% MeOH, 70° C., 4 h., 77%-82%) and the intermediate amine is acylated (1.3 equiv. of cyclopropanecarbonyl chloride (C₃H₅COCl), 1.3 equiv. of Et₃N, 0.1 equiv. of 4-DMAP, CH₂Cl₂, 25° C., 12 h., 85-95%) to afford amides 51, 41 and 55, respectively. 51, 41 and 55 are subsequently hydrolyzed (4.0 equiv. of LiOH, THF:H₂O (10:1), 25° C., 12 h., 75%-98%) to provide the mono-acids 52, 36 and 56, respectively.

Similarly, the reductive amination product of benzopyran aldehyde 60, is acylated (1.3 equiv. of cyclopropanecarbonyl chloride (C₃H₅COCl), 1.3 equiv. of Et₃N, 0.1 equiv. of 4-DMAP, CH₂Cl₂, 25° C., 12 h., 85-95%) to provide amides 53, 29 and S-7, respectively. 53, 29 and S-7 are hydrolyzed (4.0 equiv. of LiOH, THF:H₂O (10:1), 25° C., 12 h., 75%-98%) to afford the mono-acids, 54, 28 and S-8, respectively.

EXAMPLE 5 Preparation of Compounds 34, 45 and 47-49

The strategy employed for the preparation of Compounds 34, 45 and 47-49 is shown below.

Thus, S-9 is coupled by a palladium-mediated Heck reaction (2.0 equiv. of acrylonitrile or 2.0 equiv. of penta-2,4-dienoic acid methyl ester, 0.2 equiv. of Pd₂(dba)₃, 0.6 equiv. of P(o-tol)₃, 5.0 equiv. of Et₃N, DMF, 90° C., 24 h., 55% and 70% and 85%) to afford compounds 34 and 45, respectively.

Similarly, S-9 is coupled by a palladium-mediated Heck reaction (2.0 equiv. of 3-vinylbenzaldehyde, 0.2 equiv. of Pd₂(dba)₃, 0.6 equiv. of P(o-tol)₃, 5.0 equiv. of Et₃N, DMF, 90° C., 24 h., 85%) to provide the coupled benzaldehyde, which is oxidized (1.5 equiv. of NaClO₂, 4.0 equiv. of NaH₂PO₄, 10 equiv. of 2-methyl-2-butene, THF:t-BuOH:H₂O (3:1:1), 25° C., 3 h., 98%) and the resulting acid is methylated (10.0 equiv. of CH₂N₂, Et₂O, 0° C., 1 h., 100%) to provide the methyl ester 48. S-9 also undergoes a palladium-mediated Suzuki reaction (5.0 equiv. of (3-methoxycarbonylphenyl)boronic acid or (4-methoxycarbonyl-phenyl) boronic acid, 0.2 equiv. of Pd(PPh₃)₄, toluene:MeOH:1M Na₂CO₃ (10:3:1), 90° C., 24 h., 75% and 78%) to afford biphenyls 47 and 48, respectively.

EXAMPLE 6 Preparation on Compounds 30-33, 35, 37-40, 42 and 43

The strategy for the preparation of compounds 30-33, 35, 37-40, 42 and 43 is

Thus, 29 is transesterified (0.5 equiv. of n-Bu₂Sn═O, EtOH, or i-PrOH, 25° C., 48 h., 50% and 34%) to afford esters 30 and 31, respectively. 29 is reduced (1.2 equiv. of diisobutylaluminum hydride (Dibal-H), toluene, −78° C., 0.5 h., 52%) to provide the allyl alcohol 32. 32 is O-alkylated (2.0 equiv. of NaH, 3.0 equiv. of MeI, 0° C., 1 h., 95%), or is acylated (1.2 equiv. of MeOC(O)Cl, 2.0 equiv. of Et₃N, 0.1 equiv. of 4-DMAP, CH₂Cl₂, 25° C., 24 h, 90% or 1.2 equiv. of MeOC(O)Cl, 2.0 equiv. of Et₃N, 0.1 equiv. of 4-DMAP, CH₂Cl, 25° C., 24 h., 90%) to afford compounds 33, 42 and 43, respectively. 29 is hydrolyzed (4.0 equiv. of LiOH, THF:H₂O (10:1), 25° C., 24 h. 88%) to the acid which is aminated by mixed anhydride formation followed by exposure to an amine, (1.2 equiv. of EtOC(O)Cl 1.5 equiv. of Et₃N, 0.1 equiv. of 4-DMAP, CH₂Cl₂, 25° C., 1 h., then 3.0 equiv. of NH₃, MeNH₂, PhNH₂ or ((CH₂)₅)N, CH₂Cl₂, 25° C., 12 h., 85%-95%) to provide amides 37-40. 29 also undergoes cyclopropanation (10.0 equiv. of CH₂N₂, 0.2 equiv. of Pd(OAc)₂, Et₂O, 25° C., 12 h., 95%) to afford compound 35.

Representative procedures for the preparation of Region II modified compounds are shown in Examples 7 and 8.

EXAMPLE 7 Preparation of Compounds 61-77, 80-84, S-10 and S-11

The strategy employed for the preparation of 61-77, 80-84, S-10 and S-11 is shown below.

29 R = cyclopropyl 61 R = CH₃62 R = CH₂CH₃63 R = t-Bu 64 R = CF₃65 R = cyclohexyl 66 R = furyl 67 R = thiophene 76 R = 2-naphthyl

Thus, benzopyran 60 is reductively aminated (2.0 equiv. of 1-bromo-3-aminobenzene 130, THF, 70° C., 4 h., 70° C., then 2.0 equiv. of NaCNBH₃, 10% MeOH, 70° C., 4 h., 70%) and the intermediate amine coupled to methyl acrylate via a palladium mediated Heck reaction (1.5 equiv. of methyl acrylate, 0.2 equiv. of Pd₂(dba)₃, 0.5 equiv. of P(o-tol)₃, 5.0 equiv. of Et₃N, DMF, 24 h., 65%) to afford the amine S-10. S-10 is N-alkylated (5.0 equiv. of NaH, 5.0 equiv. of PhBr, PhI or MeBr, EtOH, 80° C., 70%-85%) to provide compounds 78 and 79, respectively. S-10 is also N-acylated (5.0 equiv. of an acid chloride (RCOCl), 5.0 equiv. of Et₃N, 0.2 equiv. of 4-DMAP, CH₂Cl₂, 25° C., 24 h. 55%-100%) to afford products 29, 61-77 and S-11. S-10 is further N-acylated (5.0 equiv. of an unsubstituted or substituted phenyl isocyanate RNCO, 5.0 equiv. of Et₃N, CH₂Cl₂, 25° C., 24 h. 75%-85%) to provide urea products 80 and 81, respectively. Finally, S-10 amine is N-acylated (5.0 equiv. of a substituted phenyl isothiocyanate RNCS, 5.0 equiv. of Et₃N, CH₂Cl₂, 25° C., 24 h., 50%-70%) to afford the thiourea products 83 and 84.

EXAMPLE 8 Preparation of Compounds 87, 94, 98-101, 103 and S-13

The strategy employed for the preparation of 87, 94, 98-101, 103 and S-13 is shown below.

Thus, amine S-12 is acylated (2.0 equiv. of benzoyl chloride, 2.0 equiv. of Et₃N, 0.2 equiv. of 4-DMAP, CH₂Cl₂, 25° C., 24 h., 95%) to afford benzopyran amide 103. 103 is oxidized (10 equiv. of DMDO, acetone, 0° C., 1 h., 100%) to provide epoxide S-13 (100%). S-13 undergoes ring opening (5.0 equiv. of PhSH, Amberlyst-15 catalyst, CH₂Cl₂, 25° C., 24 h., 95%) to afford the alcohol-sulfide compound S-14. S-14 is acylated (2.0 equiv. of acetic anhydride, 2.0 equiv. of Et₃N, 0.2 equiv. of 4-DMAP, CH₂Cl₂, 25° C., 24 h., 90%) to provide the acylated product, S-15. The acetate S-15 and the alcohol S-14 are coupled to methyl acrylate via a Heck reaction (2.0 equiv. of methyl acrylate, 0.2 equiv. of Pd₂(dba)₃, 0.6 equiv. of P(o-tol)₃, 5.0 equiv. of Et₃N, DMF, 90° C., 24 h., 70%-84%) to afford esters 98 and 99, respectively. Epoxide S-13 undergoes ring opening (5.0 equiv. of piperidine, CH₂Cl₂, 25° C., 24 h., 90%) to afford the alcohol-amino compound S-16. S-16 is acylated (2.0 equiv. of acetic anhydride, 2.0 equiv. of Et₃N, 0.2 equiv. of 4-DMAP, CH₂Cl₂, 25° C., 24 h., 90%) to provide the acylated product, S-17. The acetate S-17 and the alcohol S-16 are coupled to methyl acrylate via a Heck reaction (2.0 equiv. of methyl acrylate, 0.2 equiv. of Pd₂(dba)₃, 0.6 equiv. of P(o-tol)₃, 5.0 equiv. of Et₃N, DMF, 90° C., 24 h., 70%-84%) to afford esters 100 and 101, respectively. Similarly, epoxide S-13 undergoes ring opening (5.0 equiv. of H₂O, Amberlyst-15 catalyst, THF, 25° C., 48 h., 95%) to afford the diol S-18. S-18 is coupled to methyl acrylate via a Heck reaction (2.0 equiv. of methyl acrylate, 0.2 equiv. of Pd₂(dba)₃, 0.6 equiv. of P(o-tol)₃, 5.0 equiv. of Et₃N, DMF, 90° C., 24 h., 70%-84%) to provide ester 94. Epoxide S-13 also undergoes ring opening (2.0 equiv. of Et₂AICN, CH₂Cl₂, 0° C., 1 h., 83%) and elimination (40% KOH:MeOH (1:2), 25° C., 24 h., 90%) to afford the conjugated cyano compound S-19. S-19 is coupled to methyl acrylate via a Heck reaction (2.0 equiv. of methyl acrylate, 0.2 equiv. of Pd₂(dba)₃, 0.6 equiv. of P(o-tol)₃, 5.0 equiv. of Et₃N, DMF, 90° C., 24 h., 70%-84%) to provide ester 87.

Representative procedures for the preparation of Region III modified compounds are shown in Examples 9 to 17.

EXAMPLE 9 Preparation of Compounds 85, 93, 95, 102, S-20, S-21, S-22 and S-23

The strategy employed for the preparation of Compounds 85, 93, 95, 102, S-20, S-21, S-22 and S-23 is shown below.

Thus, benzopyran amide 103 is oxidized (0.02 equiv. of OsO₄, 2.0 equiv. of NMO, acetone, H₂O (10:1), 25° C., 24 h., 85%) to afford diol S-20. S-20 is diacylated (5.0 equiv. of acetic anhydride, 10.0 equiv. of Et₃N, 0.2 equiv. of 4-DMAP, CH₂Cl₂, 25° C., 24 h., 90%) to provide the diacetate S-21. The diol S-20 and the diacetate S-21 are coupled to methyl acrylate via a Heck reaction (2.0 equiv. of methyl acrylate, 0.2 equiv. of Pd₂(dba)₃, 0.6 equiv. Of P(o-tol)₃, 5.0 equiv. of Et₃N, DMF, 90° C., 24 h., 65%-80%) to afford esters 93 and 95, respectively. Intermediate benzopyran amide 103 is reduced (10% Pd/C, EtOAc, 25° C., 0.5 h., 100%) to afford amide S-22. S-22 is coupled to methyl acrylate via a Heck reaction (2.0 equiv. of methyl acrylate, 0.2 equiv. of Pd₂(dba)₃, 0.6 equiv. of P(o-tol)₃, 5.0 equiv. of Et₃N, DMF, 90° C., 24 h., 65%-80%) to provide ester 85. Intermediate benzopyran amide 103 is also cyclopropanated (CHCl3, 50% NaOH, (7:1), adogen 464 catalyst, 25° C., 6 h., 85%) to afford compound S-23. S-23 is coupled to methyl acrylate via a Heck reaction (2.0 equiv. of methyl acrylate, 0.2 equiv. of Pd₂(dba)₃, 0.6 equiv. of P(o-tol)₃, 5.0 equiv. of Et₃N, DMF, 90° C., 24 h., 65%-80%) to provide ester 102.

EXAMPLE 10 Preparation of Compounds 110, 111, 114-118, 147 and 148

The strategy employed for the preparation of Compounds 110, 111, 114-118, 147 and 148 is shown below.

Thus, 3-bromo-aniline 130 is acylated (1.1 equiv. of C₆H₁₁COCl, 1.3 equiv. of Et₃N, 0.05 equiv. of 4-DMAP, CH₂Cl₂, 25° C., 3 h., 95%) to afford amide 131. 131 is coupled to methyl acrylate via a Heck reaction (4.0 equiv. of methyl acrylate, 0.2 equiv. of Pd₂(dba)₃, 0.6 equiv. of P(o-tol)₃, 5.0 equiv. of Et₃N, DMF, 90° C., 12 h., 80%) to provide ester 132. 132 is N-alkylated (1.1 equiv. of NaH, THF, 0° C., 30 min., then 1.3 equiv. of benzyl bromides, THE, 2 h., 60%-90% where R—X=methyl iodide, benzyl bromide, 2-bromobenzyl bromide, 3-bromobenzyl bromide, 4-bromobenzyl bromide, 4-tert-butyl benzyl bromide, 3-methoxy benzyl bromide, 3,5-dimethoxy benzyl bromide, 3-(trifluoromethyl) benzyl bromide, 2-naphthyl benzyl bromide) to afford compounds 105, 110-112, 114-118 and 148.

EXAMPLE 11 Preparation of Compounds 106-109

The strategy employed for the preparation of Compounds 106-109 is shown below.

Thus, aryl bromides 114 and 115 are coupled to tert-butyl acrylate via a Heck reaction (4.0 equiv. of tert-butyl acrylate, 0.2 equiv. of Pd₂(dba)₃, 0.6 equiv. of P(o-tol)₃, 5.0 equiv. of Et₃N, DMF, 90° C., 12 h., 80%) to afford esters 109 and 110, respectively. 109 and 110 are acidified (20% TFA in CH₂Cl₂, 25° C., 1 h., 95%) to provide acids 108 and 106, respectively.

EXAMPLE 12 Preparation of Compounds 105 and 112

The strategy employed for the preparation of Compounds 105 and 112 is shown below.

Thus, 3-bromo-aniline 130 is N-acylated (1.1 equiv. of C₆H₁₁COCl, 1.3 equiv. of Et₃N, 0.05 equiv. of 4-DMAP, CH₂Cl₂, 25° C., 3 h., 95%) to afford amide 131. 131 is coupled to methyl acrylate via a Heck reaction (4.0 equiv. of methyl acrylate, 0.2 equiv. of Pd₂(dba)₃, 0.6 equiv. of P(o-tol)₃, 5.0 equiv. of Et₃N, DMF, 90° C., 12 h., 80%) to provide ester 132. 132 is N-acylated (1.1 equiv. of para-bromoC₆H₄COCl, 1.3 equiv. of Et₃N, 0.05 equiv. of 4-DMAP, CH₂Cl₂, 25° C., 3 h., 95%) to afford tertiary amide 112. 112 is coupled to tert-butyl acrylate via a Heck reaction (4.0 equiv. of tert-butyl acrylate, 0.2 equiv. of Pd₂(dba)₃, 0.6 equiv. of P(o-tol)₃, 5.0 equiv. of Et₃N, DMF, 90° C., 12 h., 80%) to provide diester 105.

EXAMPLE 13 Preparation of Compounds 121-129

The strategy employed for the preparation of Compounds 121-129 is shown below.

Thus, aryl bromide 112 is coupled to para-substituted styrene via a Heck reaction (4.0 equiv. of styrene, or para-methoxy styrene, or para tert-butyl styrene, 0.05 equiv. of Pd₂(dba)₃, 0.15 equiv. of P(o-tol)₃, 5.0 equiv. of Et₃N, DMF, 90° C., 12 h., 65%-80%) to afford esters 121, 122 and 123, respectively. 112 is coupled to unsubstituted and substituted phenyl and thiophene via a Suzuki reaction (2.5 equiv. of boronic acid, 0.2 equiv. of Pd(PPh₃)₄, toluene:MeOH: 1M Na₂CO₃ (10:3:1), 80° C., 12 h., 60%-80%) to provide compounds 124-129.

EXAMPLE 14 Preparation of Compounds 105, 133, 134, 136-138, 159, 160 and S-24

The strategy employed for the preparation of Compounds 105, 133, 134, 136-138, 159, 160 and S-24 is shown below.

Thus, para-bromobenzaldehyde is coupled to tert-butyl acrylate via a Heck reaction (4.0 equiv. of tert-butyl acrylate, 5.0 equiv. of Et₃N, 0.05 equiv. of Pd₂(dba)₃, 0.15 equiv. of P(o-tol)₃, DMF, 90° C., 12 h., 85%) to afford aldehyde S-24. S-24 is reductively aminated (1.5 equiv. of 3-bromoaniline, 0.05 equiv. of AcOH, MeOH, 25° C., 30 min., then 1.7 equiv. of NaCNBH₃, 1 h., 90%) to provide amine S-25, which is acylated (1.1 equiv. of C₆H₁₁COCl, 1.3 equiv. of Et₃N, 0.05 equiv. of 4-DMAP, CH₂Cl₂, 25° C., 3 h., 90%) to afford aryl bromide S-26. S-26 is coupled via a Heck reaction (4.0 equiv. of acrylate or 4.0 equiv. of allyl ether, 5.0 equiv. of Et₃N, 0.05 equiv. of Pd₂(dba)₃, 0.15 equiv. of P(o-tol)₃, DMF, 90° C., 12 h., 60%-85%) to provide compounds 105, 133, 134 and 136-138.

EXAMPLE 15 Preparation of Compounds 140-146 and S-28

The strategy employed for the preparation of Compounds 140-146 and S-28 is shown below.

Thus, aldehyde S-24 is coupled to amine S-27 via a reductive amination (0.05 equiv. of AcOH, MeOH, 25° C., 30 min., then 1.2 equiv. of NaCNBH₃, 25° C., 1 h., 85%) to afford amine S-28. S-28 is N-acylated (2.0 equiv. of acid chloride, 3.0 equiv. of Et₃N, 0.05 equiv. of 4-DMAP, CH₂Cl₂, 25° C., 1 h., 80%-95%) to provide compounds 105 and 140-144. S-28 is also acylated (2.0 equiv. of isocyanate, 3.0 equiv. of Et₃N, 0.05 equiv. of 4-DMAP, CH₂Cl₂, 25° C., 1 h., 60%-80%) to afford urea compounds 145 and 146.

EXAMPLE 16 Preparation of Compounds 104, 105, 139 and 150-158

The strategy employed for the preparation of Compounds 104, 105, 139 and 150-158 is shown below.

Thus, aryl bromide 112 is coupled to acrylates via a Heck reaction (4.0 equiv. of acrylate, 5.0 equiv. of Et₃N, 0.05 equiv. of Pd₂(dba)₃, 0.15 equiv. of P(o-tol)₃, DMF, 90° C., 12 h., 50%-80%) to afford compounds 105 and 150-155. Ester 105 is hydrolyzed (20% TFA in CH₂Cl₂, 1 h., 25° C., 95%) to provide acid 104. Acid 104 is esterified (1.2 equiv. of DCC, 10 equiv. of i-PrOH or BnOH, 0.2 equiv. of 4-DMAP, DMP, 25° C., 12 h., 60%) to afford compounds 152 and 153, respectively. Aryl bromide 112 is coupled to alkenes via a Heck reaction (4.0 equiv. of methyl vinyl ether, ethyl vinyl ether and phenyl vinyl ether, 5.0 equiv. of Et₃N, 0.05 equiv. of Pd₂(dba)₃, 0.15 equiv. of P(o-tol)₃, DMF, 90° C., 12 h., 50%-80%) to provide compounds 156 to 158, respectively. Further, aryl bromide 112 is reduced (0.05 equiv. of 10% Pd/C, H₂ (1 atm.), EtOAc, 25° C., 30 min., 100%) to afford the saturated ester, which is coupled to tert-butyl acrylate via a Heck reaction (4.0 equiv. of tert-butyl acrylate, 5.0 equiv. of Et₃N, 0.05 equiv. of Pd₂(dba)₃, 0.15 equiv. of P(o-tol)₃, DMF, 90° C., 12 h., 35%-75%) to provide compound 139.

EXAMPLE 17 Preparation of Compounds 161-167 and S-29

The strategy employed for the preparation of Compounds 161-167 and S-29 is shown below.

Thus, 2,4-dihydroxybenzaldehyde S-28 is selectively monoprotected (1.0 equiv. of SEM-Cl, 1.2 equiv. of Et₃N, CH₂Cl₂, 25° C., 12 h., 75%) to afford the para hydroxyl compound S-29. S-29 is O-alkylated (1.05 equiv. of Tf₂O, 1.2 equiv. of Et₃N, CH₂Cl₂, −78° C., 1 h., 95%) and the triflate is coupled to tert-butyl acrylate via a Heck reaction (4.0 equiv. of tert-butyl acrylate, 5.0 equiv. of Et₃N, 0.05 equiv. of Pd₂(dba)₃, 0.15 equiv. of P(o-tol)₃, DMF, 90° C., 12 h., 76%) to provide compound S-30. Aldehyde S-30 is coupled to amine S-27 via a reductive amination (1.2 equiv. of S-27, 0.05 equiv. of AcOH, MeOH, 25° C., 1 h., then 1.5 equiv. of NaCNBH₃, 2 h., 80%) to afford amine S-31. S-31 is N-acylated (1.2 equiv. of C₆H₁₁COCl, 1.5 equiv. of Et₃N, 0.05 equiv. of 4-DMAP, CH₂Cl₂, 25° C., 4 h., 90%) to provide amide S-32. S-32 is deprotected (3.0 equiv. of BnBr, 5.0 equiv. of K₂CO₃, DMF, 80° C., 12 h., 65%) to afford alcohol 161. 161 is alkylated with (3.0 equiv. of MeI, 5.0 equiv. of K₂CO₃, DMF, 80° C., 12 h., 90%) to provide methyl ether 162; or with (3.0 equiv. of BnBr, 5.0 equiv. of K₂CO₃, DMF, 80° C., 12 h., 65%) to afford benzyl ether 163, or acetylated with (3.0 equiv. of BrCH₂COOEt, 5.0 equiv. of K₂CO₃, DM, 80° C., 12 h., 90%) to provide 167. Alcohol 161 is O-alkylated (3.0 equiv. of AcCl, BzCl or MsCl, 5.0 equiv. of Et₃N, CH₂Cl₂, 2 h., 70%-90%) to provide compounds 164, 165 and 166, respectively.

EXAMPLE 18 Preparation of Compounds 121, 125, 126 and 174-264

The strategy employed for the preparation of Compounds 121, 125, 126 and 174-264 is shown below.

Thus, Boc protected cinnamic acid 168 is immobilized on resin (1.0 equiv. of Merrifield Resin, (0.91 mmol/mg), 2.0 equiv. of Cs₂CO₃, 0.5 equiv. of TBAI, DMF, 55° C., 24 h.) to afford resin 169. 169 is deprotected (20% TFA in CH₂Cl₂, 25° C., 1 h.) and the resultant resin-bound amine is reductively alkylated with 4-bromobenzaldehyde (10.0 equiv. of 4-aminobenzaldehyde, 0.05 equiv. of AcOH, THF:MeOH (2:1), 25° C., 1 h., then 8 equiv. of NaCNBH₃, THF:MeOH (2:1), 25° C., 2 h.) to provide amino resin 170. 170 is acylated (for R¹COCl: 30 equiv. of R¹COCl, 40.0 equiv. of Et₃N, 1.0 equiv. of 4-DMAP, CH₂Cl₂, 25° C., 12 h., for R¹NCO, 30.0 equiv. of R¹NCO, 40.0 equiv. of Et₃N, 1.0 equiv. of 4-DMAP, DMF, 65° C., 60 h.) with one of three acyl groups to afford amide or urea resins 171. The acylated resins (171) were subjected to either Heck coupling with thirteen substituted styrenes (as illustrated below; 8.0 equiv. of styrene, 10.0 equiv. of Et₃N, 0.5 equiv. of Pd₂(dba)₃, 1.5 equiv. of P(o-tol)₃, DMF, 90° C., 48 h.) or Suzuki coupling with eighteen boronic acids (as illustrated below; 5.0 equiv. of boronic acid, 3.0 equiv. of Cs₂CO₃, 0.5 equiv. of Pd(PPh₃)₄, DMF, 90° C., 24 h.) to provide stilbene resins 172 and biaryl resins 173, respectively.

Hydrolysis of these resins (172 and 173) with base (10.0 equiv. of NaOMe, Et₂O:MeOH (10:1), 25° C., 20 min.) affords compounds 121, 125, 126 and 174-264. Analysis of the library by LCMS after purification showed the average purity of these compounds to be >95%.

It will be apparent to those skilled in the art that various changes may be made in the invention without departing from the spirit and scope thereof, and therefore, the invention encompasses embodiments in addition to those specifically disclosed in the specification, but only as indicated in the appended claims. 

1. A compound having the structure:

wherein: A is a C3 up to C8 branched chain alkyl or substituted alkyl group, a C3 up to C7 cycloalkyl or substituted cycloalkyl, an optionally substituted aryl or an optionally substituted heteroaryl, X is —C(O)— or —CH₂—, R is methyl or ethyl, R¹ is H, hydroxy, alkoxy, benzoyloxy, mesityloxy, or —OCH₂C(O)OC₂H₅, R² is H or R² can cooperate with R³ to form a benzopyran, wherein the pyran ring has the structure:

wherein: R⁶ is not present if the pyran ring is unsaturated, or, if present, is selected from H, —OR, wherein R is alkyl or acyl, or R⁶ can cooperate with R⁷ to form a cyclic acetal, a cyclic ketal, or a cyclopropyl moiety, and only one of R⁷ and R⁸ is present if the pyran ring is unsaturated, or R⁷ and R⁸ are independently H, carboxyl, cyano, hydroxy, alkoxy, thioalkyl, aryl, or R⁷ and R⁸ taken together comprise a carbonyl oxygen or an oxime nitrogen, or either R⁷ or R⁸ can cooperate with R⁶ to form a cyclic acetal, a cyclic ketal, or a cyclopropyl moiety, R³ can cooperate with R² to form a benzopyran having the structure set forth above, or R³ is alkenyl, optionally substituted aryl or heteroaryl, or optionally substituted arylalkenyl or heteroarylalkenyl, R⁴ is H or hydroxy, and R⁵ is H, hydroxy, alkoxy or aryloxy.
 2. The compound of claim 1 wherein R² and R³ cooperate to form a benzopyran.
 3. The compound of claim 2 wherein A is cyclopropyl, X is —C(O)—, R¹ is methoxy, R⁶ and R⁷ are absent, and R⁴, R⁵ and R⁸ are hydrogen.
 4. The compound of claim 2 wherein A is cyclopropyl, X is —CH₂—, R¹ is methoxy, R⁶ and R⁷ are absent, and R⁴, R⁵ and R⁸ are hydrogen.
 5. The compound of claim 2 wherein A is cyclohexyl, X is —C(O)—, R¹ is methoxy, R⁶ and R⁷ are absent, and R⁴, R⁵ and R⁸ are hydrogen.
 6. The compound of claim 2 wherein A is phenyl, X is —C(O)—, R¹ is methoxy, R⁶ and R⁷ are absent, and R⁴, R⁵ and R⁸ are hydrogen.
 7. The compound of claim 2 wherein A is phenyl, X is —C(O)—, R¹ is methoxy, R⁶ and R⁷ cooperate to form a dichlorocyclopropyl ring, and R⁴, R⁵ and R⁸ are hydrogen.
 8. The compound of claim 2 wherein A is cyclohexyl, X is —C(O)—, R¹ is methoxy, R⁶ and R⁷ cooperate to form a dichlorocyclopropyl ring, and R⁴, R⁵ and R⁸ are hydrogen.
 9. The compound of claim 1 wherein R³ is alkenyl.
 10. The compound of claim 9 wherein A is cyclohexyl, X is —C(O)—, R¹ R², R⁴ and R⁵ are hydrogen, and R³ is —CH═CH—C(O)—O-tBu.
 11. The compound of claim 1 wherein R³ is optionally substituted aryl or heteroaryl.
 12. The compound of claim 11 wherein said compound is selected from the group consisting of compounds wherein: A is cyclohexyl, X is —C(O)—, R¹, R², R⁴ and R⁵ are each hydrogen, and R³ is selected from the group consisting of phenyl, p-thiomethyl-phenyl, m-methoxy-phenyl, m-acetyl-phenyl, 5-methyl-2-thiophene-yl, 5-acetyl-2-thiophene-yl, 4-dimethylamino-phenyl and 2,3-(O—CH₂—O)-phenyl. 13.-18. (canceled)
 19. The compound of claim 11 wherein said compound is selected from the group consisting of compounds wherein: A is isopropyl, X is —C(O)—, R¹, R², R⁴ and R⁵ are each hydrogen, and R³ is 4-dimethylamino-phenyl or 2,3-(O—CH₂—O)-phenyl. 20.-21. (canceled)
 22. The compound of claim 1 wherein R³ is or optionally substituted arylalkenyl or heteroarylalkenyl.
 23. The compound of claim 22 wherein said compound is selected from the group consisting of compounds wherein: A is cyclohexyl, X is —C(O)—, R¹, R², R⁴ and R⁵ are each hydrogen, and R³ is selected from the group consisting of —CH═CH-phenyl, —CH═CH-p-methoxy-phenyl, —CH═CH-o-fluoro-phenyl, —CH═CH-m-fluoro-phenyl, and —CH═CH-p-fluoro-phenyl.
 24. The compound of claim 22 wherein said compound is selected from the group consisting of compounds wherein: A is isopropyl, X is —C(O)—, R¹, R², R⁴ and R⁵ are each hydrogen, and R³ is selected from the group consisting of —CH═CH-phenyl, —CH═CH-o-fluoro-phenyl, —CH═CH-m-fluoro-phenyl, and —CH═CH-p-fluoro-phenyl. 25.-31. (canceled)
 32. A formulation comprising at least one compound according to claim 1 in a pharmaceutically acceptable carrier therefor. 